Helminths of the black-headed gull (Chroicocephalus ridibundus) from breeding colonies in north-central Poland

Among parasites of the digestive tract of the black-headed gull (Chroicocephalus ridibundus) in Poland, the best known are species of digenetic trematodes and cestodes. Nematodes of this bird species are not well known. Black-headed gulls, due to their varied diet, migration, life in a flock, and changes of habitat, can become infected with various species of helminths, and like synanthropic birds, they can spread the dispersal stages of parasites across urban and recreational areas. In the present study, an attempt was made to identify the helminth fauna of C. ridibundus from breeding colonies in north-central Poland. The aim of the study was to describe the taxonomic structure of parasites of the digestive tract of the black-headed gull and determine the quantitative parameters of their occurrence. A total of 43 black-headed gulls were examined post-mortem for gastrointestinal helminths, resulting in the identification of four cestodes (Paricterotaenia porosa, Lateriporus clerci, Anomotaenia micracantha, and Wardium fusum), three trematodes (Diplostomum pseudospathaceum, Plagiorchis laricola, and Apophallus muehlingi), and three nematodes (Eucoleus contortus, Cosmocephalus obvelatus, and Porrocaecum ensicaudatum). Lateriporus clerci (in adult form), C. obvelatus and P. ensicaudatum (in larval form) were recorded for the first time in the black-headed gull in Poland.


Materials and methods
The material for the research consisted of nematodes, trematodes and cestodes isolated from the gastrointestinal tracts of 36 adult black-headed gulls.Parasitological examination was performed in 2017-2019 on 43 blackheaded gulls found dead in breeding colonies situated in north-central Poland, in the Kuyavian-Pomeranian Voivodeship, on small islands in lakes Kusowskie, Koronowskie, and Pakoskie, as well as on the River Brda flowing through Bydgoszcz and on an island in the active gravel pit Skoki Duże, a Natura 2000 protected area for birds.A detailed description of the islands and the size of the colonies are presented in works by Kitowski et al. 18 , Jakubas et al. 19 and Indykiewicz et al. 20 .Parasites were identified using standard methods, including fixation, staining, and preparation of microscope slides.Cestodes were fixed in 75% ethyl alcohol, and trematodes and nematodes in 70% ethyl alcohol.Cestodes were stained with iron-acetocarmine prepared according to the formula given by Georgiev 21 .Slides of selected cestode specimens were prepared in Hoyer's medium 22 .Trematodes were stained with carmine alum, cleared in clove oil, and mounted in Canada balsam 23,24 .Unfixed nematode specimens cleared in glycerine were examined under a microscope 24 .
Helminth species were identified using available keys and numerous original works.The taxonomic affiliation of cestodes was presented according to Pojmańska and Cielecka 25 and Pojmańska et al. 1 .Species identification of trematodes was carried out using keys given by Niewiadomska 26 , Niewiadomska and Pojmańska 27 , Yamaguti 28,29 , Gibson et al. 30 , Jones et al. 31 , and Bray et al. 32 , as well as original works by Bychovskaja-Pavlovskaja 33 , Niewiadomska 34 , and Sonin 35 .The taxonomic affiliation of nematodes was determined according to Baruš and Sergeeva 36 , Stapf et al. 37 , Kim et al. 3 , McNeill and Anderson 39,40 and Smogorzhevskaya 41 .The taxonomic affiliation of 17 cestode and 24 trematode specimens was not determined, and 39 nematode specimens were identified only to subfamily.This was due to severe damage to the bodies of the parasites, most likely resulting from the substantial degree of decomposition of several of the birds.Quantitative parameters of the occurrence of parasites, i.e. prevalence and intensity of infection (mean intensity and range of intensity), were calculated according to definitions given by Bush et al. 42 In this study, prevalence refers to the percentage of infected gulls among all tested gulls; mean intensity refers to the number of parasites divided by the number of infected gulls; and range of intensity refers to the minimum and maximum number of parasites infecting a single gull.
A statement confirming the permission for collecting the dead birds:
There were 26 black-headed gulls infected with nematodes.The isolated nematodes were assigned to three species: Eucoleus contortus (Creplin, 1839), Cosmocephalus obvelatus (Creplin, 1825) Seurat, 1919, and Porrocaecum ensicaudatum (Zeder, 1800) (Table 1).Nematodes of the species E. contortus and those identified to the subfamily Capillariinae spp.colonized the oesophagus of the hosts.C. obvelatus was isolated from the proventriculus.All P. ensicaudatum specimens were in the third larval stage and located under the stratum corneum at the site where the proventriculus passes into the gizzard.The first, highly characteristic morphological character of the larvae, making them easy to spot on the background of the dissected gizzard, is a black intestine visible through the colourless body.The anterior segment of the body is rounded.The three labia around the mouth typical of sexually mature forms are absent.However, there is a smooth ring of cuticle, visible on the optical microscope slide as two globules situated on each side of the anterior end (Fig. 1).In some specimens, a cap of arcadial tissue was observed below the mouth.Inside the body of the larvae, an oesophagus and a ventriculus could be seen.Their dimensions and those of other parts of the body are presented in Table 2.The posterior end of the body terminates in a conical tail (Fig. 2).There are no male or female sex organs.

Discussion
Among the helminth fauna of the black-headed gull, nematodes were the group with the highest prevalence, followed by cestodes, while the fewest birds were infected with trematodes.This pattern of prevalence is in agreement with the findings of Indykiewicz and Janiak 4 .This is the first record of an adult specimen of the species Lateriporus clerci (Johnston, 1912) in Poland.Gulls are definitive hosts of this parasite, while amphipod crustaceans of the genus Gammarus function as intermediate hosts 25 .The life cycle of the cestode may also include ducks as a paratenic host.It has been recorded in Europe and Asia, e.g. in C. ridibundus, Larus canus, and Larus argentatus ssp.mongolicus from Lake Baikał in southeastern Siberia, as well as in Chroicocephalus philadelphia from Cooking Lake in Alberta, Canada 43,44 .In Poland, Czapliński et al. 45 detected larvae of this cestode in Gammarus pulex in the Masurian Lake District.
The black-headed gull is a definitive host for all cestode species recorded in the study.The species identified in the research material had previously been recorded (except for L. clerci) among the cestode fauna of these birds in Poland and other parts of Europe 1,46,47 .The material was not confirmed to include cestodes of the genus Hymenolepis, whose eggs were previously found in 60 adult black-headed gulls from breeding colonies on an island in a lake in Myślęcinek, in the Kuyavian-Pomeranian Voivodeship 4 .
Paricterotaenia porosa (syn Choanotaenia porosa Rudolphi, 1810) is a widespread parasite in Europe, Asia, and North and Central America.Mature specimens have been recorded in the black-headed gull, common gull, great black-backed gull, and common tern.The intermediate host in its life cycle is unknown 25 .In Poland, cestodes of this species have been identified in gulls on the Baltic Coast 48,49 , in the Masurian Lake District 50,51 , in the Wielkopolsko-Kujawska Lowland 52,53 , and in eastern Poland 54 .In Europe, P. porosa has been recorded in Chroicocephalus ridibundus in Denmark (in 16 gulls among 111 examined) 55 and in northern Karelia in Russia  5 .In Great Britain, cestodes identified only to genus as Paricterotaenia sp. were detected only in a single five-day-old black-headed gull chick 56 .Gulls and terns function as definitive hosts of Anomotaenia micracantha (syn Anomotaenia micracantha micracantha Krabbe, 1869).Its intermediate host is unknown.It has been recorded in Europe and North America 25 .Cestodes of this species have been recorded in Poland in the black-headed gull and black tern in the Masurian Lake District 50 and the Wielkopolsko-Kujawska Lowland 53 .
Gulls are a definitive host of Wardium fusum, syn.Aploparaksis fusus (Krabbe, 1869), and the intermediate host is the crustacean Artemia salina 25 .The species has been recorded in Europe and Asia, and in Poland in the black-headed gull on the Baltic Coast 48,49 and in the Wielkopolsko-Kujawska Lowland 53 .
The black-headed gull is a host for all species of trematodes recorded in the study.Trematodes are common parasites of black-headed gull, recorded earlier in this bird in Poland, with broad host specificity 1 .Diplostomum pseudospathaceum, Plagiorchis laricola and Apophallus muehlingi are associated through their life cycles with freshwater environments.The fact that these trematodes were recorded in a synanthropic environment in the region of the Vistula is indicative of foraging by gulls in the inland ecosystem and of a diet consisting in part of natural food which included the hosts of trematodes, which were present in them in larval form (metacercariae).These include fish, insect larvae, crustaceans, and molluscs.This finding indirectly demonstrates the occurrence of invertebrates in the foraging grounds of gulls-snails of the genera Lymnaea, Stagnicola, and Lithoglyphus, which in the life cycles of trematodes are hosts of parthenogenetic forms of trematodes.The three trematode species identified in the material have also been recorded in black-headed gulls in the Czech Republic 7 .In northern Karelia in Russia, as well as in Great Britain and Denmark, the occurrence of other trematode species belonging to the same genera as those recorded in the present study has been described 5,55,56 .   1 stress that some published data on Diplostomum spathaceum may in fact refer to D. pseudospathaceum, as in the past these two species were not distinguished 34 .Cercariae of D. pseudospathaceum develop in the great pond snail Lymnaea stagnalis and the marsh pond snail Stagnicola palustris.Metacercariae are found in the eye lens of many species of fish, including cyprinids and percids 26 .Diplostomum spathaceum has often been recorded in fish in Poland, but according to Niewiadomska 61 , it is a species complex which includes D. pseudospathaceum.It is difficult to distinguish Diplostomum species on the basis of the morphology and morphology of metacercariae due to their close similarity and individual variation.Adult forms of these trematodes do not exhibit such high variation of taxonomic characters as the metacercariae.In a study by Morozińska-Gogol 62 , in order to determine the Diplostomum species found in three-spined stickleback Gasterosteus aculeatus on the Baltic coast, black-headed gulls were experimentally infected with these metacercariae.The adult trematodes obtained in this manner were predominantly identified as D. spathaceum, indicating that this trematode is the most numerous parasite of the eye of three-spined stickleback in the Baltic 62 .
Plagiorchis laricola is a widespread species and is found in many species of birds, including storks, plovers and gulls.The host for the sporocysts and cercariae are the great pond snail and the wandering snail Radix balthica, and the metacercariae develop in insect larvae, crustaceans, and molluscs 27 .
Apophallus muehlingi is a parasite of piscivorous birds.In Poland it has been recorded in gulls of the genus Larus and Gavia stellata.It has also been found in some mammals that feed on fish 1 .The host of the rediae and mother sporocysts is the gravel snail Lithoglyphus naticoides.The metacercariae develop in cyprinid fish 63 .The role of the gravel snail as a host is confirmed by the results of research and analyses by numerous authors [64][65][66][67] .In Poland, larval forms of A. muehlingi occurring in gastropods have not been described.The gravel snail is an alien species in Poland, from the Pontic-Caspian steppe region.At the same time it is one of the endangered species listed in the Polish Red Book of Animals.Its occurrence has been described at many river sites in Poland, including the Vistula 68,69 .In the light of current knowledge of the role of L. naticoides in the life cycle of the trematode A. muehlingi, this snail can be said to contribute to the persistence of the A. muehlingi population in the area where the gulls for the research were obtained.
The presence of two nematode species has thus far been recorded in Poland: Cyathostoma lari and Eucoleus contortus 1 .The present study confirmed the occurrence of E. contortus (subfamily Capillariinae), and additionally nematodes Cosmocephalus obvelatus and Porrocaecum ensicaudatum were detected.The presence of C. obvelatus is the first recorded case in the gull C. ridibundus in Poland.This species is found in gulls and other birds associated with the aquatic environment (e.g.Mergus serrator, Sterna arctica, Gavia arctica, Podiceps cristatus, and Alca torda) 38,41,70 .Adult forms parasitize the oesophagus or proventriculus of the definitive host.The life cycle includes an intermediate host-invertebrates of the order Amphipoda (genera Gammarus, Hyalella, and Crangonyx) 71 .In Poland, the presence of this nematode species has been described in Phalacrocorax carbo sinensis 1 .Porrocaecum ensicaudatum in the form of third-stage larvae is the first confirmed case in the black-headed gull in Poland.Sexually mature nematodes parasitize birds of the order Passeriformes.They develop to the invasive L3 larval form in the intermediate host, which is earthworms 39 .In birds of the order Charadriiformes (the genus Larus), the development of L3 larvae is inhibited.Birds of the species C. ridibundus are considered to be atypical hosts.P. ensicaudatum larvae live under the stratum corneum at the site where the proventriculus passes into the gizzard.A morphologically similar species for which the black-headed gull and other birds of the order Charadriiformes are recognized as definitive hosts is P. semiteres.Both of these nematode species can parasitize the definitive host in the invasive larval stage.The two species can be distinguished by calculating the ratio of the ventriculus length to the intestinal caecum length 70,72,73 .The length of the caecum of P. ensicaudatum larvae is no more than half the length of the ventriculus 74 .Analysis of measurements of the length of the intestinal caecum and ventriculus reveals a ratio of 1:0.29-0.35.The results are similar to the values obtained by Supryaga and Supryaga 75 .
E. contortus had the highest prevalence and intensity among all recorded nematodes.Indykiewicz and Janiak 4 also reported a predominance of nematode eggs of the genus Capillaria (subfamily Capillariine) in the droppings of black-headed gulls in comparison to other groups of helminths.Infection of gulls with nematodes E. contortus confirms the presence of earthworms in the diet of these birds.Earthworms (Oligocheta) are intermediate hosts of nematodes of the genera Capillaria and Eucoleus 37,70 .The black-headed gull is a typical host for the nematode species found in the present study.Their presence has been described in the black-headed gull in countries such as the Czech Republic 73 and Great Britain 76 .
As colonial birds, gulls have higher species richness of parasitic worms than non-colonial birds 77 .The species richness of the helminth fauna of birds of the order Charadriiformes is primarily determined by the habitat (freshwater and/or marine) and diet (omnivory, invertebrates, fish-based diet) 78 .Black-headed gulls are hosts for nearly 200 species of parasitic worms, with trematodes believed to have the highest species richness (120 species detected) (London Natural History Museum host-parasite database 79 ).This is confirmed by research by Gutiérrez et al. 78 .The authors noted that the species richness of trematodes is higher in birds whose diet includes mixed food sources (invertebrates and fish) or in omnivorous birds occupying marine or freshwater/marine habitats.The number of species of nematodes, cestodes and acanthocephalans is not influenced by the host diet or habitat, but in the case of nematodes, they predominate over other helminths in the parasite fauna of birds which are habitat generalists.The number of trematode species also increases with geographic range, while the species richness of cestodes increases with geographic latitude.The species richness of nematodes decreases as migration distance increases 78 .
The helminth fauna of black-headed gulls C. ridibundus from several areas of Russia, i.e. northern Karelia, the Republic of Mordovia, and Lake Baikal in south-eastern Siberia, has been studied during the last 10 years 5,44,80 .Dorzhiev et al. 44 examined nearly the same number of gulls as in the present study.While the total prevalence of helminths in black-headed gulls from Lake Baikal was 79.5%, in the present study it was more than 83%.There are considerable differences in the number of species of parasitic worms.Gulls from Lake Baikal were infected with 11 species of cestodes, 23 species of trematodes, 10 species of nematodes, and 3 species of acanthocephalans.In comparison with these data, the species richness of parasites of black-headed gulls from breeding colonies in north-central Poland seems quite modest: 4 species of cestodes, 3 species each of trematodes and nematodes, and no acanthocephalans.However, the species composition of helminths in gulls from these regions overlaps to only a small extent, mainly in the case of cestodes 44 .No nematodes were found in gulls from the Republic of Mordovia in Russia, but the birds were infected with two species of cestodes, which were also found in the material in the present study, and 4 species of trematodes, of which one, Plagiorchis laricola, was present in gulls from Poland.The helminth fauna of black-headed gulls from North Karelia was poorer in species than in the material analysed in the present study.A species common to both regions was Paricterotaenia porosa.
Other Laridae species in which similar species richness of helminths has been described are the European herring gull Larus argentatus and Larus argentatus ssp.Mongolicus 44,79,81,82 .The abundance of parasite species can be explained by the range of occurrence of these birds and their type of diet.In seagulls from both the Kola Bay of the Barents Sea and Lake Baikal, trematodes were represented in the highest numbers, with fewer cestodes and nematodes 44,81,82 .
Gulls Chroicocephalus ridibundus, like Larus argentatus, can be hosts of cestodes Diphyllobothrium dendriticum, which pose a threat to human health.Infections with D. dendriticum in humans still receive little attention, due to their sporadic occurrence.However, they are an example of a food-borne zoonosis and a consequence of the globalization of the fish trade, transport of fresh fish on ice, increased human migration, and changes in culinary habits.Recent cases of infection with D. dendriticum in the Netherlands, Switzerland and the Czech Republic indicate the need to address factors spreading parasites which are rare or do not belong to the native fauna 83 .The gulls mentioned above can also be hosts of the cestode Ligula intestinalis, which parasitizes cyprinid fish in the form of plerocercoid larvae 44,[83][84][85] .This parasite does not pose a threat to humans.
Helminth species were precisely identified in the present study, but there were certain limitations, e.g. the poor state of some of the carcasses.Nevertheless, in addition to the fresh carcasses examined, birds that were not fresh were used as well, and it was possible to obtain helminths from them in accordance with literature recommendations [86][87][88][89] .In future endeavours, the use of molecular methods in addition to morphological methods would be of value.Molecular methods can be useful in studies of cryptic species.For example, the use of genetic methods has proven useful in identifying the cryptic diversity of Diplostomum species, including species identification of specimens obtained from intermediate and definitive hosts [90][91][92][93] .Researchers studying Plagiorchis sp.provide molecular evidence of the high diversity of the genus in snails and stress the need to use molecular methods in further study of this diversity, as well as the importance of further molecular testing to establish the links between various stages in the life cycle of these parasites 94,95

Conclusion
The results of the present study contribute new information regarding helminths of the black-headed gull.
Given the new information about the parasite species found in black-headed gull and the dependency of their occurrence on the diet of these birds and their foraging grounds, parasitological research should be continued.

Figure 1 .
Figure 1.The anterior end of the body of the Porrocaecum ensicaudatum larva.The arrows indicate the ring of cuticle, visible on the optical microscope slide as two globules situated on each side of the anterior end.

Figure 2 .
Figure 2. The posterior end of the body of the Porrocaecum ensicaudatum larva.The arrow indicates the conical tail.

Table 1 .
Prevalence and intensity of helminth infection in black-headed gulls.